The abuse potential of cocaine, amphetamine and related psychostimulants result from a direct interaction of these substances with the Na+/CI- dependent biogenic amine transporters. These transporters mediate rapid reuptake of biogenic amines from the synaptic cleft and include the transporters for dopamine (DAT), norepinephrine and serotonin. The long term goal of the present proposal is to understand the molecular and cellular mechanisms governing the activity and availability of this class of transporters in the presynaptic membrane and how this is modulated by endogenous substrate and by drugs of abuse. Insight into these mechanisms has a strong potential of unrevealing new therapeutical strategies for both drug abuse and psychiatric disorders. In this grant we propose to test the hypothesis that the C-terminus of the DAT serves multiple distinct roles in the regulation of endoplasmic reticulum (ER) export, targeting, internalization, recycling, degradation, and phosphorylation of the transporter. The extreme C-terminus of DAT contains a type 2 PDZ (PSD-95/Discs-large/ZO-1 homology) binding sequence shown to interact with the PDZ domain protein PICK1. Deletion of this PDZ binding sequence (Leu-Lys-Val) leads to retention of DAT in the ER, and it has been assumed, therefore, that interactions with PDZ proteins are critical for ER export and surface targeting. However, based on analysis of C-terminal DAT mutations, we have shown that although the extreme C-terminus of the DAT is indispensable for ER export and surface targeting, these processes do not appear to require PDZ domain interactions. Instead we surmise a key role of PDZ domain interactions for regulating transporter trafficking and phosphorylation as well as a role of non-PDZ mediated interactions for ER export. We will test these hypotheses by utilizing the unique ensemble of expertises available in the PPG group. Moreover, we will continue to develop and apply advanced fluorescence technologies in the proposed studies both within this component and in collaboration with the other components of the PPG.